Monitoring Domoic Acid in Marine Phytoplankton by Disposable Immunosensors

Advances in sensor technology have allowed the significant progress in the monitoring of noxious compounds in the sea, providing real-time detection so as to prevent risks associated with the diffusion and dispersion of toxic substances in the environment. An important element in the overall picture is the harmful algal blooms which pose serious threats to marine ecosystems through the production of toxins that accumulate in filter-feeders and ultimately impact both human health and fisheries. Domoic acid is a neurotoxic amino acid produced by marine planktonic diatoms of the genus Pseudo-nitzschia. Here we monitored domoic acid production by natural Pseudo-nitzschia populations in phytoplankton samples collected along the Middle Tyrrhenian coast, over the course of one year, using selective immunosensors based on screenprinted electrodes, using differential pulse voltammetry as the electrochemical technique, to yield quantitative outputs. In this work, disposable devices have been applied for monitoring the production of domoic acid on algal extracts and the results have been validated by conventional high pressure liquid chromatography-ultraviolet detection methods. The data obtained revealed the presence of domoic acid in Italian phytoplankton, especially in coastal impacted areas, highlighting the potential risk of toxin entering into marine food webs and the environment. Immunosensors based on screen-printed electrodes prove to be effective tools for annual monitoring of domoic acid in seawater samples, thus providing a reliable early warning system relative to health and economic impact of algal toxins.

A Surface Plasmon Resonance-based Immunosensors for Sensitive Detection of Heroin

A simple technique for sensitive detection of heroine based on surface- plasmonresonance has been theoretically and experimentally investigated. The experiment was realized by using an anti-MO monoclonal antibody and a morphine (MO)-bovine serum albumin (MOBSA) conjugate (antigen). The reason for using MO-BSA in the detection of heroine was also discussed. MO-BSA was immobilized on a gold thin film of SPR sensor chip by physical adsorption. The configuration of the device is allowed to be further miniaturized, which is required for the construction of a portable SPR device in the application of in-situ analysis.

Electrochemical Immunosensors for the Diagnosis of Celiac Disease

Celiac disease is a permanent intolerance to gluten proteins of wheat, rye, barley, and oats in genetically susceptible individuals. The clinical picture is characterized by inflammation and damage of the small intestinal mucosa and malabsorption of essential nutrients. Therapeutically, a lifelong strict gluten-free diet is necessary. The diagnosis of celiac disease is complex and includes symptomatology, serology, small intestinal histology, and genetic status. Serological testing plays a central role within the diagnostic procedure and is based on the measurement of disease-specific antibodies against gluten proteins (antigen) and tissue transglutaminase (autoantigen). Immunofluorescence detection and enzyme-linked immunosorbent assays are currently most often applied for antibody testing. However, these tests are expensive and time-consuming. Therefore, simple and rapid alternative methods have been developed during the last years, and electro-chemical immunosensors seem to be the most promising analytical tools. The architecture of these sensors may comprise the following elements: working and reference electrodes, covalent or noncovalent binding of the antigen to the surface of the working electrode by means of a functional monolayer, and blocking of unreacted binding sites. The analytical procedure is initiated by adding the analyte (serum antibodies) and an analyte-specific second antibody, which is usually labeled with an enzyme. The special reaction of the enzyme with an appropriate substrate results in a product that initiates a current that can be measured by different electrical methods. A number of different electrochemical immunosensors variable in different electrodes, binding systems, secondary antibodies, and current measurements have been developed. Most of them have been tested with real human serum samples of celiac patients and healthy individuals, and some of them reached disease sensitivity and specificity comparable with traditional analytical systems. Thus, electrochemical immunosensors can be promising alternatives to existing diagnostic tests in the future. They are simple, reliable, robust, user-friendly, and cost-effective tools with short operation times.

Electrochemical and colorimetric dual-signal detection of Staphylococcus aureus enterotoxin B based on AuPt bimetallic nanoparticles loaded Fe-N-C single atom nanocomposite

Sensitive detection of Staphylococcus aureus enterotoxin B(SEB)is of importance for preventing food poisoning from threatening human health.In this work,an electrochemical and colorimetric dual-signal detection assay of SEB was developed.The probe(Ab2/AuPt@Fe-N-C)was bound to SEB captured by Ab1,where the Ab2/AuPt@Fe-N-C triggered methylene blue degradation and resulted in the decrease of electrochemical signal.Furthermore,the probe catalyzed the oxidation of 3,3’,5,5’-tetramethyl biphenyl to generate a colorimetric absorbance at 652 nm.Once the target was captured and formed a sandwich-like complex,the color changed from colorless to blue.SEB detection by colorimetric and electrochemical methods showed a linear relationship in the concentration ranges of 0.0002-10.0000 and 0.0005-10.0000 ng/mL,with limits of detection of 0.0667 and 0.1670 pg/mL,respectively.The dual-signal biosensor was successfully used to detect SEB in milk and water samples,which has great potential in toxin detection in food and the environment.

Glucose oxidase as a blocking agent-based signal amplification strategy for the fabrication of label-free amperometric immunosensors

An effective electrochemical signal amplification strategy based on enzyme membrane modification and redox probe immobilization was proposed to construct an amperometric immunosensor.L-cysteine@ferrocene functionalized chitosan,which possessed not only efficient redox-activity but also excellent film-forming ability,was coated on the bare glass carbon electrode. Moreover,the thiol groups(SH)in the ferrocenyl compound were used for gold nanoparticles immobilization via the strong bonding interaction,which could further be utilized for the immobilization of antibody biomolecules with well-retained bioactivities.Finally,glucose oxidase(GOD)as the enzyme membrane was employed to block the possible remaining active sites and avoid the nonspecific adsorption.With the excellent electrocatalytic properties of GOD towards glucose,the amplification of antigen-antibody interaction and the enhanced sensitivity could be achieved.Under the optimal conditions,the linear range of the proposed immunosensor for the determination of carcinoembryonic antigen(CEA)was from 0.05 to 100 ng/mL with a detection limit of 0.02 ng/mL(S/N=3).Moreover,the immunosensor exhibited good selectivity,stability and reproducibility, which provided a promising potential for clinical immunoassay.

Highly sensitive and selective electrochemical immunosensors by substrate-enhanced electroless deposition of metal nanoparticles onto three-dimensional graphene@Ni foams

In this study, we have for the first time preformed the facile substrate-enhanced electroless deposition(SEED) of metal nanoparticles onto monolithic graphene@Ni foams for construction of disposable three-dimensional(3 D) electrochemical immunosensors. Specifically, we firstly used the SEED method to deposit gold nanoparticles(AuNPs) onto the graphene@Ni foam for immobilization of antibody(Ab1). This is followed by a second step SEED deposition to produce silver nanoparticles(AgNPs) for electrochemical stripping detection. Using a-fetoprotein antigen(AFP) as a module analyte, the newly-developed sensor showed a wide linear response, ranging from 5.0 pg/mL to 5.0 ng/mL and a low detection limit down to 2.3 pg/mL. The newly-developed 3 D-immunosensor is sensitive, reliable,and easy to be fabricated, showing great potential for clinic applications.

Diagnostic tests for hepatitis C: Recent trends in electrochemical immunosensor and genosensor analysis

Hepatitis C is a liver disease that is transmitted through contact with the blood of an infected person. An estimated 150 million individuals worldwide have been chronically infected with the hepatitis C virus(HCV). Hepatitis C shows significant genetic variation in the global population, due to the high rate of viral RNA mutation. There are six variants of the virus(HCV genotypes 1, 2, 3, 4, 5, and 6), with 15 recorded subtypes that vary in prevalence across different regions of the world. A variety of devices are used to diagnose hepatitis C, including HCV antibody test, HCV viral load test, HCV genotype test and liver biopsy. Rapid, inexpensive, sensitive, and robust analytical devices are therefore essential for effective diagnosis and monitoring of disease treatment. This review provides an overview of current electrochemical immunosensor and genosensortechnologies employed in HCV detection.There are a limited number of publications showing electrochemical biosensors being used for the detection of HCV.Due to their simplicity,specificity,and reliability,electrochemical biosensor devices have potential clinical applications in several viral infections.

A novel impedance immunosensor based on O-phenylenediamine modified gold electrode to analyze abscisic acid

An impedance immunosensor based on O-phenylenediamine modified gold electrode for the determination of phytohormone abscisic acid(ABA) was proposed.The operating pH,absorption time,absorption temperature and concentration of anti-ABA antibody were investigated to optimize the analytical performance.The calibration curve for the determination of ABA was obtained from this impedance immunosensor under optimal conditions.The results showed that the detection limit at about 1 ng/mL in the range of 10-5000 ng/mL...

A Novel Immunosensor Based on Au Nanoparticles and Polyaniline/Multiwall Carbon Nanotubes/Chitosan Nanocomposite Film Functionalized Interface

A novel multilayer film based on Au nanoparticles(AuNPs) and polyaniline/carboxylated multiwall carbon nanotubes-chitosan nanocomposite(PANI/MWCNTs/CS) was exploited to fabricate a highly sensitive immunosensor for detecting chlorpyrifos. PANI-coated MWCNTs were prepared by in situ chemical polymerization and carboxylated MWCNTs played an important role in obtaining the thin and uniform coating of PANI resulting in the improved immunosensor response. Au NPs were used as a linker to immobilize chlorpyrifos antibody. The performance of the immunosensor was characterized by means of cyclic voltammetry(CV), electrochemical impedance spectroscopy(EIS) and scanning electron microscopy(SEM), respectively. All variables involved in the preparation process and analytical performance of the immunosensor were optimized.Under optimal conditions(antibody concentration: 5 μg/mL, working buffer pH: 6.5, incubation time: 40 min,incubation temperature: 25℃), the immunosensor exhibited a wide linear range from 0.1 to 40× 10^(-6)mg/mL and from 40 × 10^(-6)mg/mL to 500 × 10^(-6)mg/mL, and with a detection limit of 0.06 × 10^(-6)mg/mL, which provided a valuable tool for the chlorpyrifos detection in real samples.

Recent advances in quantum dots-based biosensors for antibiotics detection

Antibiotics are a category of chemical compounds used to treat bacterial infections and are widely applied in cultivation,animal husbandry,aquaculture,and pharmacy.Currently,residual antibiotics and their metabolites pose a potential risk of allergic reactions,bacterial resistance,and increased cancer incidence.Residual antibiotics and the resulting bacterial antibiotic resistance have been recognized as a global challenge that has attracted increasing attention.Therefore,monitoring antibiotics is a critical way to limit the ecological risks from antibiotic pollution.Accordingly,it is desirable to devise new analytical platforms to achieve efficient antibiotic detection with excellent sensitivity and specificity.Quantum dots(QDs)are regarded as an ideal material for use in the development of antibiotic detection biosensors.In this review,we characterize different types of QDs,such as silicon,chalcogenide,carbon,and other doped QDs,and summarize the trends in QD-based antibiotic detection.QD-based sensing applications are classified according to their recognition strategies,including molecularly imprinted polymers(MIPs),aptamers,and immunosensors.We discuss the advantages of QD-derived antibiotic sensors,including low cost,good sensitivity,excellent stability,and fast response,and illustrate the current challenges in this field.

Noninvasive Label-Free Detection of Cortisol and Lactate Using Graphene Embedded Screen-Printed Electrode

A sensitive and specific immunosensor for the detection of the hormones cortisol and lactate in human or animal biological fluids, such as sweat and saliva, was devised using the label-free electrochemical chronoamperometric technique. By using these fluids instead of blood,the biosensor becomes noninvasive and is less stressful to the end user, who may be a small child or a farm animal.Electroreduced graphene oxide(e-RGO) was used as a synergistic platform for signal amplification and template for bioconjugation for the sensing mechanism on a screenprinted electrode. The cortisol and lactate antibodies were bioconjugated to the e-RGO using covalent carbodiimide chemistry. Label-free electrochemical chronoamperometric detection was used to analyze the response to the desired biomolecules over the wide detection range. A detection limit of 0.1 ng mL^(-1) for cortisol and 0.1 mM for lactate was established and a correlation between concentration and current was observed. A portable, handheld potentiostat assembled with Bluetooth communication and battery operation enables the developed system for point-of-care applications. A sandwich-like structure containing the sensing mechanisms as a prototype was designed to secure the biosensor to skin and use capillary action to draw sweat or other fluids toward the sensing mechanism. Overall, the immunosensor shows remarkable specificity, sensitivity as well as the noninvasive and point-of-care capabilities and allows the biosensor to be used as a versatile sensing platform in both developed and developing countries.

A MXene-functionalized paper-based electrochemical immunosensor for label-free detection of cardiac troponin I

Convenient,rapid,and accurate detection of cardiac troponin I(cTnI)is crucial in early diagnosis of acute myocardial infarction(AMI).A paper-based electrochemical immunosensor is a promising choice in this field,because of the flexibility,porosity,and cost-efficacy of the paper.However,paper is poor in electronic conductivity and surface functionality.Herein,we report a paper-based electrochemical immunosensor for the label-free detection of cTnI with the working electrode modified by MXene(Ti_(3)C_(2))nanosheets.In order to immobilize the bio-receptor(anti-cTnI)on the MXene-modified working electrode,the MXene nanosheets were functionalized by aminosilane,and the functionalized MXene was immobilized onto the surface of the working electrode through Nafion.The large surface area of the MXene nanosheets facilitates the immobilization of antibodies,and the excellent conductivity facilitates the electron transfer between the electrochemical species and the underlying electrode surface.As a result,the paper-based immunosensor could detect cTnI within a wide range of 5-100 ng/mL with a detection limit of 0.58 ng/mL.The immunosensor also shows outstanding selectivity and good repeatability.Our MXene-modified paper-based electrochemical immunosensor enables fast and sensitive detection of cTnI,which may be used in real-time and cost-efficient monitoring of AMI diseases in clinics.

A Micro Amperometric Immunosensor Based on MEMS

A micro amperometric immunosensor with the sensitive area of only 1mm^2 was fabricated on silicon using the technique of Micro-Electro-Mechanical Systems (MEMS).A double exposure of SU-8 photoresist process was developed to create both the sensitive pool and reaction pool.Antibody was immobilized via cross-linking with glutaraldehyde on the sensitive area of the electrode surface,which was electropolymerized with polypyrrole previously.The immunosensor was characterized by detection of human immunoglobulin G (HIgG).The immunosensor displayed a good linear response to HIgG concentrations between 5ng/ml and 255ng/ml and demonstrated a fast response time of 3 minutes.

Development of a QCM (Quartz Crystal Microbalance) Biosensor to the Detection of Aflatoxin B1

In this study, we have used a direct immunoassay where the simple binding between antigen and an antibody is detected. Immunoassays were performed in a drop system, monitoring the frequency decrease of the quartz-crystal microbalance device because of mass increasing during immunoreaction. The QCM sensor was coated on both sides by gold electrodes, only one side of the crystal (liquid side) was in contact with the solution;the other side (contact side) was always dry. We tested a piezoelectric immunosensor for aflatoxin B1 (AFLA-B1) mycotoxin detection through the immo- bilization of DSP-anti-AFLAB1 antibody (AFLA-B1-Ab anti AFLAB1) on gold-coated quartz crystals (AT-cut/5 MHz). The DSP (3,3’-Dithiodipropionic-acid-di-N-hydroxysuccinimide ester) was used for the covalent attachment of the proteins. The piezoelectric crystal electrodes were pretreated by DSP for 15 min, rinsed with water and dried in a gentle flow of nitrogen gas. Then the DSP-coated crystals were installed in a sample holder and exposed to the anti-AFLAB1 antibody and to the AFLA-BI. Frequency and resistance shifts (Δf and ΔR) were measured simultaneously. Δf versus AFLA-BI concentrations in the range of 0.5 - 10 ppb exhibited a perfect linear correlation with a coefficient of above 0.998.

Immunosensor Based on Surface Plasmon Resonance for Antigen Recognition

A novel immunosensor based on surface plasmon resonance（SPR） has been developed for the recognition of antigen. The sensor was designed on the basis of the fixed angle of incidence and measuring the reflected intensities in a wavelength range of 430--750 nm in real-time. An ultra-bright white light-emitting diode（LED） was used as the light source. Molecular self-assembling in solution was used to form the sensing membrane on gold substrate. It has been seen that the sensitivity of the SPR sensor with 3-mercaptopropionic acid（MPA）/protein A（SPA） sensing membrane is considerably higher than that with MPA or SPA modified sensing membrane. The kinetic processes on the sensing membrane were studied. The human B factor（Bf）, an activator of complement 3（C3）, was recognized among the other antigens. This sensor can also be used for other antigen/antibody or adaptor/receptor recognition. Under optimized experimental conditions, the sensor has good selectivity, repeatability, and reversibility.

DEVELOPMENT OF A VERSATILE SENSING MEMBRANE FOR IMMUBILIZATION OF PROTEIN AND A NEW IMMOBILIZATION METHOD

ＤＥＶＥＬＯＰＭＥＮＴＯＦＡＶＥＲＳＡＴＩＬＥＳＥＮＳＩＮＧＭＥＭＢＲＡＮＥＦＯＲＩＭＭＵＢＩＬＩＺＡＴＩＯＮＯＦＰＲＯＴＥＩＮＡＮＤＡＮＥＷＩＭＭＯＢＩＬＩＺＡＴＩＯＮＭＥＴＨＯＤＹ．Ｋ．Ｚｈｏｕ，Ｊ．Ｗ．Ｙｕａｎ，Ｓ．Ｑ．Ｘｕ，Ｂ．Ｈ．Ｓｈｕ（Ｄ...

Studies on the Voltammetric Determination of Clenbuterol at One-touch Immunochips

A novel immunosensor for determination of clenbuterol was described in this paper. Horseradish peroxidase (HRP) and Monoclonal antibody against clenbuterol was covalently immobilized onto the cellulose acetate membrane which was used as one-touch immunochips. After 20 min of competitive reaction of clenbuterol in samples against glucose oxidase-conjugated clenbuterol (CLB-GOD) with anti-clenbuteol antibody immobilized on the membrane, the one-touch transducer was fixed on the Nafion - ferrocene modified glass-carbon electrode (GCE, working electrode) by “O” ring. The increased current changes (ΔI) were recorded for the quantitative determination of clenbuterol by derivative cyclic voltammetry. Experimental results showed that a very effective system for electron transmission had been established using dual-enzyme system and Nafion-ferrocene modified GCE. Quantitative analysis of clenbuterol could be easily performed on this kind of immunosensor, which showed the advantage of time-saving and high sensitivity (The detecting limit of this kind of immunosensor can be down to 100 ng/L). In addition, other conditions affecting the electrochemical response were also discussed in detail.

A MEMS Based Amperometric Immunosensor with Self-assembled Monolayers Immobilization Technique

Based on MEMS technology,immunosensor with an'Au,Pt,Pt'three-microelectrode system enclosed in a SU-8 micro pool was fabricated.Employing SAMs technique,the Au electrode was modified by cysteamine(Cys)to assemble gold nanopanicles(nanogold)layer,subsequently,a layer of protein G(PG)was immobilized on nanogold layer to further capture antibody orientedly.Compared with the immunosensors using bulky gold electrode and direct PG binding to electrode immobilization technique for antibody,it has attractive advantages,such as miniaturization,good compatibility,broad linear range for human immunoglobulin(HIgG)and easy to be designed into array.

Experimental Study of A Novel Piezoelectric Tumor Marker Micro-array Immunosensor

A novel 2×5 model of insert-plug piezoelectric quartz crystal tumor marker micro-array immunosensor constructed with screw clamp apparatus has been developed for quantitative detection of the tumor markers such as alpha-fetoprotein (AFP),carcino-embryonic antigen (CEA),prostate specific antigen (PSA),and human chorionic gonadotropin (hCG) in serum,in which every crystal unit can oscillate independently with the stability of±1 hertz (Hz) in air and±2 Hz in liquid.These response characteristics of Pz tumor marker micro-array immunosensor such as temperature, time-cost,reproducibility and specificity etc were also investigated.The detection ranges for AFP,CEA,PSA,and hCG obtained by Pz micro-array immunosensor were 20 ng/ml～640 ng/ml,1.56 ng/ml～50 ng/ml,1.25 ng/ml～50 ng/ml,and 2.5 mIU/ml～250 mlU/mi respectively with the coefficient of variance (CV) less than 5%.No cross-reactivates with other tumor markers in serum were observed.The results of AFP,CEA,PSA,and hCG obtained by this method from 68 serum samples were in good agreement with those given by chemiluminescence immunoassay with the correlation coefficients of 0.92,0.90,0.91,and 0.94 respectively.The Pz immunosensor regenerated by urea solution could be reused for five times without appreciable loss of response activity.Therefore,the proposed insert-plug immunosensor provides a rapid, sensitive,specific,reusable,convenient and reliable alternative for the detection of tumor markers in clinical laboratory.

Amplified Electrochemical Immunoassay Using HRP Labeled Protein as an Inhibitor to Silver Deposition in the Presence of H_2O_2

A novel electrochemical immunosensor with amplification effect based on the enzyme inhibition of silver deposition was proposed. In this method, the capture antibody was first immobilized onto a gold electrode via a self-assembled layer. After a sandwich immunoreaction, HRP labeled antibody was bound to the gold electrode. The HRP on the electrode inhibited silver deposition when the electrode was incubated in hydroquinone-H2O2 solution and silver ion solution. The linear sweep voltammetry was chosen to detect the deposited silver and the result showed that the peak current was linearly proportional to the content of IgG in the range of 50 to 2500 ng/mL with a detection limit of 35 ng/mL.